Can the molar extinction coefficient for NADPH vary?

[maddensd]

Hi There,

 

I am optimising an enzyme assay that measures the appearance of NADPH. I began by creating a standard curve ([NADPH] (mM) vs. ABS@340nm) for NADPH in the buffer that I intend to use to identify my lower and upper detection limits. I performed it in triplicate and got nice tight data (all STDEV were less than 10% of each point - in most cases less than 1%). The linear portion of the graph has an R2 value of 0.9981 (5 data points). The only problem is that the slope is only 4.98, but it should by around 6.022, as this would be the correct millimolar extinction coefficient for NADPH. Can variables like temperature or buffer composition change the extinction coefficient of a compound? Should I be worried if the slope is wrong, considering everything else looks well on the graph?

 

Shane.

[qed]

well , the extinction is, as far as i remember, not that temperature sensitive, the buffer composition has a greater effect on the coefficient.

 

Is it possible that your absolute extinction values were too high and therefor out of the linear range of the photometer? For common facilities the linear range ends about absorbtion values of 1,5.

[angelkissesH]

I beleive I've read somewhere (lecture notes from the internet so not exactly from a good source) that NADP+ doesn't absorb at 340nm. Based on the buffer solution, could it be possible that some of you NADPH may have converted to NADP+ thus decreasing the conc of NADPH (and the extinction coefficient)?